The inner retina releases a lot of lactate, consistent with the high lactate concentration (3.8 - 13 mM) at the SRS even in light-adapted eyes. Lactate is released upon dark-adaptationa result of (1) an increased glucose metabolism at the outer retina, (2) the reduced retinal oxygen level in the dark adapted eye and (3) glutamate-induced lactate release from Mller cells. To prevent lactate accumulation at the SRS, the RPE expresses lactate transporters, of the MCT family of monocarboxylate transporters, at the apical (MCT1) and basolateral membrane (MCT3 and MCT4). To demonstrate the importance of lactate transport in the eye, the same group also showed that mice lacking MCT1, MCT3, and MCT4 expression rapidly lose photoreceptor cells. Lactate transport was also shown to increase fluid transport across the porcine RPE, and in the bullfrog, possibly by interacting with other ion-transporters. A recent study by Becker and colleagues demonstrated that MCT1 activity increases when the electrogenic Na/HCO3 co-transporter (NBC1) is co-expressed with MCT1. In addition, MCT1 activity is enhanced by carbonic anhydrase activity in muscle cells. Therefore, a similar functional protein-complex may exist in vivo in the RPE. Our study aims to determine if MCT mediated lactate-transport involve CAs, Na/HCO3 co-transporter or other ion-transporters in the RPE by monitoring intracellular pH, transepithelial potential (TEP), and total tissue resistance (Rt). [unreadable] [unreadable] Lactate transport across the apical membrane of the RPE is a two phase process; when lactate is perfused onto the apical membrane, it causes a fast intracellular acidification (phase 1), followed by a slow alkalinization (phase 2). These pH-responses are mediated by monocarboxylate transporters because perfusing lactate, pyruvate, propionate, or acetate to the apical bath caused similar pH-responses. We were also able to inhibit the acidification phase (phase 1) with MCT1-inhibitors: niflumic acid and pCMBS. The recovery phase (phase 2) was blocked by amiloride and ouabain, indicating that proton-efflux via the Na/H exchanger mediates the recovery phase. Perfusing lactate onto the apical membrane causes a TEP-rise due to an increased Cl-efflux from the basolateral membrane of the RPE. We confirm this in the cultured hfRPE by showing that the apical lactate-induced TEP-response was weakened by Cl-channel inhibitor (DIDS) at the basolateral membrane. [unreadable] [unreadable] In the RPE, vacuolar V-type H+ATPases are localized at the apical membrane and is actively pumping protons out of the RPE; this was evidenced by the strong intracellular acidification upon apical membrane exposure to H+ATPase inhibitors (phloretin, NBD-Cl, DCCD, and NEM). Thus the H+ATPase may mediate the pH-recovery phase (phase 2) of the lactate-response, and at the same time causes a TEP-rise. In that regard, our experiments show that phloretin partially inhibited the pH-recovery phase and the TEP-rise. [unreadable] [unreadable] A DIDS-inhibitable Na/2HCO3 co-transporter was detected at the apical membrane of the cultured hfRPE, thus corroborating earlier experiments performed in frog and bovine RPE. Our experiments show that blocking pNBC1 activity (with DIDS) did not affect H/Lac entry via MCT1, suggesting that HCO3-influx via pNBC1 was buffering H/Lac co-transport activity and that MCT1 transport activity was independent of pNBC1 activity. Since interactions between MCT1 and the cytosolic CA-II have been reported in muscle cells, we test this protein interaction in the RPE by inhibiting CA activity with dorzolamide. In the presence of dorzolamide and CO2/HCO3-rich buffer, we show that the apical lactate acidification was amplified. This indicates that H/Lac transport via MCT1 was not inhibited by dorzolamide, instead dorzolamide inhibited HCO3-entry (via pNBC1) that was buffering H/Lac transport, thus a stronger acidification was observed. [unreadable] [unreadable] Lactate transport at the basolateral membrane is mediated by MCT3 and MCT4 in cultured hfRPE cells. Interestingly, perfusing lactate to the basolateral membrane caused a slow intracellular alkalinization but a fast TEP-drop. Perfusing any monocarboxylates (i.e., lactate, acetate, pyruvate, and propionate) to the basal bath caused slow intracellular alkalinization, thus confirming that the alkalinization was a two part process: (1) acidification mediated by MCT3 H/Lac co-transport that is overwhelmed by the (2) alkalinization caused by proton efflux from MCT1, Na/H exchanger, or the H+ATPase. Blocking proton efflux from the Na/H exchanger at the apical membrane with amiloride did not reverse basal lactate induced alkalinization. However, in the presence of H+ATPase inhibitors (pCMBS, phloretin, or NBD-Cl) at the apical bath, perfusing lactate to the basal membrane caused acidification. Therefore our data suggests that proton-flux out of the apical membrane via H+ATPase and H/Lac transport out of the RPE via MCT1 caused the 20 mM basal lactate induced alkalinization.